The major goal of this project is to characterize cell surface antigens and their soluble or cell associated ligands which play critical roles in cell-cell interactions and in the interactions of T cells with their environment. We have focused our efforts on two large superfamilies of membrane antigens: 1) the integrins and 2) the Ly-6 antigens. We had previously shown that a subpopulation of immature thymocytes express a constitutively activated form of the integrin, a4b1, which appeared to mediate their adhesion to fibronectin (FN) or VCAM-1. To determine the physiological importance of this observation, immunohistochemical and confocal microscopy techniques were used to localize the expression of VCAM-1 and FN in situ in the thymus. VCAM-1 was selectively expressed by the thymic epithelium in the cortex and corticomedullary junction, while FN was expressed predominantly in the thymic medulla. These results suggest that VCAM-1 is the exclusive ligand for the binding of activated a4b1 on immature thymocytes during early stages of development in the cortex and raised the possibility that a4b1 may play a role in the survival of thymocytes during development. It was found that co-stimulation of thymocytes with plate-bound anti-TCR antibodies and FN or VCAM-1 markedly protected human CD4+CD8+ thymocytes from steroid induced apoptosis. This protection against apoptosis was associated with an increase in bcl-2 protein levels. Thus, a4b1 may play a role in thymocyte development by elevating levels of bcl-2 in T cells with relatively low affinity TCRs and protecting them from steroid induced programmed cell death. High levels of the expression of the Ly-6E antigen were shown to be directly correlated with enhanced tumorigenicity and metastatic potential of several distinct tumor lines of different histologic types. Microenvironmental factors were shown to regulate Ly-6E expression and may contribute to the generation of high or low expressing tumors which differ in tumorigenicity. It remains to be determined whether the Ly-6 antigen itself directly contributes to this process or whether Ly-6 expression is causally linked to an as yet unidentified factor which regulates the high malignancy phenotype. Another distinct member of the Ly-6 family, the TSA-1 antigen, which is widely expressed on resting B cells and activated T cells was shown to play a modulatory role in the activation of T cells by stimulatory mAbs. We demonstrated that engagement of TSA-1 functionally inhibited the crosslinking of the stimulatory mAb by the FcgRIIB (CD32) and that a physical association existed between the TSA-1 and the FcgRIIB. The mechanism of this inhibitory effect remains to be studied. Taken together, these studies on two distinct members of the Ly-6 family strongly suggest that this family of antigens is likely to interact with other cell surface antigens and/or signal transduction molecules and characterization of potential Ly-6 ligands is a priority for future studies.